2.11. Immunohistochemistry

For immunohistochemistry, one full‐thickness placental tissue section was placed in 10% buffered formalin for 12–24 hr before embedding in paraffin wax, according to standard procedures and cut into serial of 5 μm sections and put on super frosted glass slides (Micro slides, Santa Cruz Biotechnologies). Tissue sections were incubated overnight for dewaxing. Antigen retrieval was done by heating slides for 2 min in tris‐buffer saline (TBS). After drying, slides were washed with phosphate‐buffered saline (PBS) and incubated for 1 hour with incubation solution (0.05% bovine serum albumin, 0.01% Triton X and 10% goat serum). Again, slides were washed with PBS and then incubated with specific rabbit polyclonal primary antibody against eNOS (Catalogue no. ab‐5589; Abcam Biotechnology, Inc., Cambridge, United Kingdom) for 48 hr at 4°C. After incubation, slides were washed with PBS and incubated with Goat Anti‐Rabbit IgG (Alexa Fluor® 488) antibody (Catalogue no. ab‐150077; Abcam Biotechnology, Inc.) for 2 hr. Slides were then washed with PBS and kept for drying, mount with mounting medium and observed under fluorescent microscope (Bx51, Olympus, Tokyo, Japan).